Recently, enzymes have been widely used as industrial catalysts in view of the substrate specificity, reaction specificity, and stereochemical specificity thereof and mild reaction conditions. Such enzymatic catalysts, however, are limited to hydrolases which do not require an energy source, such as amylase hydrolyzing starch into saccharides, and L-amino acid acylase converting N-acyl-L-amino acid into L-amino acid.
Biosyntheses in vivo are carried out by synthetase utilizing mainly adenosine 5'-triphosphate (ATP) as an energy source, i.e., a chemical energy which is liberated when ATP is hydrolyzed into adenosine 5'-diphosphate (ADP) and orthophosphoric acid, or adenosine 5'-monophosphate (AMP) and pyrophosphoric acid. In order to extend industrial utilization of enzymes, therefore, it has been attempted to develop a new production system, generally called a "bioreactor", in which the same biosyntheses as in vivo are performed in vitro by fixing such synthetase or oxidoreductase and supplying ATP and the like. In the bioreactor system, a large amount of ATP is required as an energy source, and it is furthermore necessary to reproduce the ATP from the ADP or AMP.
For the reproduction of ATP from ADP or AMP, a method utilizing an enzyme, viz., adenylate kinase, catalyzing the following reaction is known, as described, for example, in Enzyme Engineering, Vol. 2, pp. 209 and 217 (1974). ##STR1##
From (1)+(2).times.2, the following equation becomes possible. EQU AMP+2 Acetyl phosphate.fwdarw.ATP+2 Acetic acid (3)
That is, even though ATP is converted into ADP or AMP in the bioreactor system, the reproduction of ATP becomes possible by the combination (eq. (2) or (3)) of these enzymes.
In the practical utilization of such reactions, it is required for these enzymes to be stable and sufficiently durable for industrial applications. The heretofore known adenylate kinase, however, is a very unstable enzyme which is obtained from yeast and from muscles of animals. The acetate kinase, which is obtainable from Escherichia coli, is also very unstable, which has long been known.
More recently, however, Japanese Patent Application (OPI) No. 25088/77 (the term "OPI" as used herein refers to a "published unexamined Japanese patent application") disclosed that heat-resistant acetate kinase can be collected from a thermophilic Bacillus stearothermophilus. Thus, it has been desired to obtain adenylate kinase having high stability such as that for the acetate kinase described in Japanese Patent Application (OPI) No. 25088/77.
In commercially synthesizing compounds by the use of these enzymes, it is very difficult in practice to recover the enzyme after the reaction for the reuse thereof and to effect a continuous enzymatic reaction. In order to overcome such disadvantages, various investigations have been made, and several proposals have been made. One such proposal is to fix an enzyme on a water-insoluble carrier by a suitable method, to make the enzyme water-insoluble so that it can be used repeatedly. For instance, Immobilized Enzyme by Oskar Zaborsky, CRC Press (1973) and Koteika Koso (Immobilized Enzyme) by Ichiro Chibata, Kodansha (1975) give examples of immobilizing aminoacylase with ion exchange resin by adsorption, immobilizing dextranase with cellulose by covalent bonding, and immobilizing aspartase with polyacrylamide gel by entrapping. Such immobilization of enzymes is a typical method of permitting enzymes to be used repeatedly and continuously.
For example, it has been proposed to effect immobilization for the commercial development of acetate kinase (see Hakko to Kygyo (Fermentation and Industry), Vol. 35, No. 1, pp. 3-10 (1977)). It has, however, been found that when a coliform enzyme is immobilized with cyanogen bromide according to Whitesides, et al., Enzyme Engineering, Vol. 2, p. 217 (1974), edited by E. Kendall et al., Prenum Press, the residual ratio of activity (expressed as a %) is only several % in the absence of a stabilizer, the long term stability is very poor, and the immobilized enzyme is not suitable at all for the commercial utilization thereof.
The factors inhibiting commercial utilization of enzymes includes the fact that almost all enzymes are very unstable, as well as the problem of water-solubility. Although examples in which immobilization increases the stability of the catalytic function are known, examples are also known in which the stability of the catalytic function is reduced. For example, I. Chibata concluded that, as a result of comparative examinations of the stability of enzyme before and after immobilization, that there could not be found any regularity between a method of immobilization and the stability of the immobilized enzyme, and therefore that it is very difficult to predict which method can increase the stability of enzyme (see I. Chibata, Koteika Koso (Immobilized Enzyme), p. 107, Kodansha, Tokyo (1975). It is also known that in some cases, the properties which an enzyme has originally before the immobilization are lost by the immobilization, and the specificity is changed by the immobilization (see Hakko to Kogyo (Fermentation and Industry), Vol. 35, p. 7 (1977)).
The properties of an enzyme result from the complicated and higher-order structure thereof. The higher-order structure of the enzyme is naturally influenced by the immobilization. Therefore, in order to know if a stable immobilized enzyme can be obtained by the immobilization, it is necessary to determine this by trial and error with each enzyme.
The molecular structure of the enzyme varies depending on the type of a microorganism producing the enzyme, and, furthermore, this complicated molecular structure easily changes depending on the circumstances under which the enzymatic protein is placed. Therefore, it is difficult for one skilled in the art to predict the properties, such as specificity, catalytic activity, and long term stability, of the enzyme after immobilization from the properties of the enzyme before immobilization. Therefore, when immobilization is performed using an enzyme of a different species even though the enzyme falls within the same category, it has not been possible to apply the immobilization method and conditions of the enzyme of the different species. In order to obtain an immobilized enzyme having a high residual ratio of activity and long term stability, therefore, it has been necessary to conduct investigations by the method of trial and error for each enzyme. For these reasons, it has long been desired to develop a method which permits the determination of a suitable immobilization method and immobilization conditions without relying on a trial and error method.
In the case of adenylate kinase, it has also been found that when it is to be immobilized for the commercial utilization thereof, the immobilization method and immobilization conditions must also be determined by the method of trial and error. This has seriously inhibited the immobilization of adenylate kinase and commercial utilization thereof. In fact, little study on the immobilization of adenylate kinase has been made. As one of a limited number of methods which have heretofore been reported, there is known, for example, a method in which adenylate kinase of pig muscle is immobilized in a polyacrylamide gel containing therein an N-hydroxysuccinimido group (see Methods in Enzymology, Vol. 44, p. 887 (1976), edited by K. Mosbach, Academic Press). In accordance with this known method, a complicated and delicate procedure is required so as not to deteriorate the activity of the enzyme, i.e., in the course of immobilization, adenylate kinase is added to a non-oxidizing atmosphere for several seconds until the acrylamide is solidified. The immobilization of adenylate kinase and application thereof have been subjected to significant limitations. In addition, adenylate kinase of pig muscle which cannot be immobilized on a carrier at the immobilization procedure usually loses the activity thereof, to the extent that it is practically impossible to recover and reuse. In accordance with these conventional methods, therefore, the immobilization of adenylate kinase and the production of adenine cofactors utilizing the immobilized adenylate kinase have not been practically possible. Even if adenylate kinase is immobilized irrespective of such operational and economical disadvantages, when the immobilization is performed at room temperature (20.degree. to 30.degree. C.), the activity is substantially lost, and, therefore, the immobilization should be performed at low temperature, e.g., near 0.degree. C. In this respect, the production of immobilized adenylate kinase composite has been unfeasible from the viewpoint of operationability and economics.